Summary: Developing T lymphocytes undergo a complex series of maturational events within the post-natal thymus, but relatively few of the essential signals the thymus provides have been identified. Different progenitor stages stratify in different regions of the thymus, suggesting that each region may provide a distinct signaling microenvironment. However, other than a few distinctions between cortex and medulla, the differences between various stromal microenvironments in the thymus remain enigmatic. In this proposal, we describe a method for high-throughput analysis of stromal gene expression in the thymus, with special emphasis on gene products that may provide signals to lymphoid cells in a regionally distinct manner. The approach involves micro-dissection of six functionally and anatomically distinct tissue regions, followed by transcriptional analysis of each region using high density microarrays. Genes expressed in each region will represent a combination by both lymphoid cells and stromal cells, but lymphoid genes will be filtered out using microarray results from purified lymphoid cells. We show preliminary evidence from one region, the sub-capsular cortex, that this approach results in lists of genes including many already known to be characteristic of stromal cells, as well as some not previously known to be expressed in the thymus, but with known roles in relevant biological functions (differentiation, proliferation, survival, etc.). Multiple approaches for validating the relevance of the results are described, including confirming the presence of corresponding receptors on lymphoid cells. This project will define, on a large scale, the differences between anatomically and functionally defined microenvironments in the thymus, including insights into how each region supports various stages of lymphoid differentiation, as well as into the genetic nature of the stromal cells themselves. This project thus conforms ideally to the R21 funding mechanism, using advanced technology to build hypotheses that may be formally extended to conventional (R01) funding mechanisms. Relevance: failure to make new T cells results in mild to severe immunodeficiency secondary to events such as aging or environmental exposure (radiation, viruses, chemicals). Understanding how T cells are made in the thymus is thus of direct relevance to human health, as well as being of academic interest. [unreadable] [unreadable]